Reduced ASF1 levels suppressed in vitro and in vivo AML growth and leukemia burden using human cells. (A) A schematic representation of a doxycycline-inducible shRNA model targeting ASF1A, ASF1B, or both ASF1A and ASF1B in MOLM-14 cells. (B) MOLM-14 cells were treated with 1 μg/mL doxycycline to induce knockdown for 6 days. The effect of the knockdown was measured by immunoblotting analysis. shRNA expressing cells were kept in doxycycline-containing media throughout the culture. (C) Cell viability of MOLM-14 cells expressing shASF1A, shASF1B, or both was measured by colorimetric (MTS) assays after 6 days of doxycycline-induced knockdown. (D) Cell growth over time of MOLM-14 cells expressing shASF1A, shASF1B, or both was indicated by cell counts. (E) Cell cycle analysis of MOLM-14 cells expressing shASF1A, shASF1B, or both using propidium iodide and flow cytometry after 3 days of doxycycline-induced knockdown. (F) Colony formation assay was assessed by plating MOLM-14 cells expressing shASF1A, shASF1B, or both in the methylcellulose-based medium following doxycycline-induced knockdown for 14 days. (G) MOLM-14 cells expressing shASF1A, shASF1B, or both were xenografted into sublethally irradiated NSG mice, which were randomly divided into 2 groups (n = 4-5 per group) with or without 625 mg/kg doxycycline chow. All the mice were euthanized 18 days after treatment. (H-I) The leukemic burden was determined by GFP and/or RFP positivity, human CD13/CD33, and CD15 positivity using flow cytometry. (J) Spleen weight and representative images of the spleens are shown from each group. Data are expressed as means ± SEM and the Student t tests were used for statistical analyses. ∗∗P < .01 and ∗∗∗P < .001.